Sporadic cancer is a complex disease, a consequence of the interaction of individual susceptibility with a lifetime of exposures to both nutritive and toxic compounds. The stem cell theory of cancer postulates that aberrations in stem cell self renewal and differentiation pathways are integral in carcinogenesis. Most in vitro cancer studies are undertaken in cancer cells expanded from a clonal population or in cell lines that have adapted to culture conditions. These studies often do not account for interindividual variability or previous exposures and may be limited in their ability to produce physiologically relevant data about chemopreventive nutrient treatment. Here, we propose a novel in vitro model to understand the transcriptomic effects of treatment of two compounds that have been shown to affect stem cell self renewal: the dietary polyphenol curcumin and the alkaloid piperine. We have previously used the mammosphere model to show that both curcumin and piperine specifically target breast stem cells and synergistically inhibit self-renewal while remaining non-toxic to normal differentiated cells. In Specific Aim 1, we will culture primary breast cells as well as cancer cell lines in anchorage independent conditions, and treat with curcumin and piperine, both individually and in combination. We will extract RNA for deep sequencing using the barcode analysis by sequencing method (Bar-seq). Bioinformatic analyses will indentify differences in mechanism of action of curcumin and piperine in cancer cells and normal primary breast cells. Differentially expressed genes identified in Specific Aim 1 will be validated in an expanded primary tissue and breast cancer cell line sample set in Specific Aim 2. Our application of this novel in vitro methodology will provide a crucial link in understanding the mechanism by which curcumin and piperine specifically target stem cells as well as differences in mechanisms of action in normal vs. tumor cells. Additionally, this study wil provide stem cell specific biomarkers of efficacy of treatment for curcumin and piperine that will be extended to future clinical studies. These data will provide foundational evidence as to whether nutritional compounds, such as curcumin and piperine, can be used as an intervention in breast cancer prevention in susceptible populations. PUBLIC HEALTH RELEVANCE: Sporadic breast cancer is a disease that involves the interaction between individual susceptibility and a lifetime of exposure to toxicants and nutritive compounds. In this study, we propose to develop a new in vitro model to understand how nutrients have cancer preventive properties in normal breast stem cells. Results from this study could identify methods to tell if nutritive compounds are effective treatments in future clinical studies and identify areas of the genome that are important in cancer prevention